PROJECT SUMMARY Effective vaccines remain elusive for many deadly diseases; therefore, it is critical that we better understand how the immune system generates a robust, neutralizing antibody response to vaccination so that we may enhance this type of response in future vaccine design. B cells are the producers of high-affinity antibodies, however, it is the CD4+ T cells that provide the cytokines and co-stimulatory molecules necessary to drive this B cell fate and establish long-term humoral immunity. This is why it is critical that we better understand the development and function of specific CD4+ T cell subsets involved in generating this type of response. After a primary infection or vaccination, some activated CD4+ T cells become a specialized subset specifically known to provide direct B cell help: T follicular helper (Tfh) cells. What commits T cells to the Tfh cell fate is still unknown. Our novel approach leverages the CD4+ T cell response against the immunodominant LLO epitope from Listeria in B6 mice, using two defined CD4+ TCR transgenic lines and polyclonal T cells. The two naive T cells differ in their tonic signaling mediated through the TCR recognition of self-pMHC. Naive CD4+ T cells with low tonic signaling have a high basal metabolism, respond robustly in a primary in vivo response, and develop into Tfh and TEM cells. Conversely, naive CD4+ T cells with high tonic signaling have a low basal metabolism, and poorly form Tfh cells. The premise of this proposal is that the strength of TCR:self-pMHC reactivity (tonic signaling) is deterministic for establishing the basal metabolism and the subsequent Tfh response. In Aim 1, we will establish whether a direct relationship exists between tonic signaling and the development of Tfh following antigen exposure. To this end, we will use our novel knock-in mouse line, Scn5a+. Expression of th Scn5a voltage gated sodium channel allows us to increase tonic signaling in CD4+ T cells independent of TCR signaling. We will decrease tonic signaling using a newly developed conditional knockout allele of H-2DM. Tfh helper function will be tested using an NP-LLO model. In Aim 2 we will examine how T cell metabolism influences Tfh cell responses. We have identified the glycerol phosphate shuttle as a key player in the increased metabolism of the LLO-118 T cells. We have now generated a mouse with a conditional knockout allele of mGPD2 which will be a powerful reagent to explore the role of this metabolic pathway in LLO-118 and polyclonal CD4+ T cell responses. These findings will deepen our understanding of Tfh development and may reveal therapeutic targets for vaccine design and autoimmunity.